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Formation of new blood and lymphatic vessels, called angio- and lymphangiogenesis, is essential for the development and dissemination of malignant tumors, including diffuse large B-cell lymphomas (DLBCL). It was demonstrated that the main lymphangiogenesis-involved cytokines:VEGF-C,-D and their receptor VEGFR-3 are expressed in the majority of DLBCL cases. Our previous studies revealed that VEGF-D serum concentration has a negative impact on the overall survival of DLBCL patients, however the influence of lymphangiogenesis on the clinical picture and treatment outcome assessed directly by a mean vascular density (MVD) and mean lymphatic vessels density (LVD)  has not been studied so far.

The aim of our study was to evaluate the angiogenesis and lymphangiogenesis in DLBCL lymph nodes in relationship to the clinical features and the serum VEGF-C,-D, VEGFR-3 and bFGF levels at diagnosis, in order to assess the predictive value of those cytokines as to the complete remission (CR) achievement after standard R-CHOP treatment.

The study was performed in 31 (21 M, 10 F) DLBCL patients NOS type, aged 26-84 yrs (mean 67,5 + 13,6) from the Holycross Cancer Center, Kielce, Poland.  14 patients were classified as GCB, and the remaining 17 were non-GCB. For each patient blood count, serum LDH activity, b-2microglobulin, albumin and CRP concentrations were determined. The clinical stage of the disease and the general condition were determined according to the Ann Arbor classification, ECOG and the IPI staging system. The patients received 6-8 courses of the standard 21-R-CHOP regimen, and 10 of them achieved CR, in one case no data on remission were not obtainable. The cytokines serum concentration was established by ELISA method. The angio- and lymphangiogenesis were assessed in the DLBCL lymph nodes by immunohistochemical staining with anti-CD34 and D2-40 (a marker of lymphatic endothelium) antibodies. MVD and LVD was estimated in three selected hot spots by two independent investigators. As a control, MVD and LVD in a group of 11 inflammatory lymph nodes were determined. Statistical analysis was performed using  Mann-Whitney test and the Spearman rank correlation coefficient. The p value less than 0.05 was assumed as statistically significant.

For the whole group of patients, the mean MVD and LVD were 126,7+51,1/mm² and 99,6+ 81.6/mm², respectively. For the reactive lymph nodes the respective values were 197.1+ 43,0/mm² and 84.4+19,2/mm². MVD was significantly higher in reactive than in neoplastic lymph nodes (p = 0.002). There was also a significant difference in MVD between complete as opposed to not complete remission patients (144.0+37.6/mm² and 117.9+56.7/mm²;p=0.03). We did not find any relationship between LVD and the clinical parameters studied. MVD displayed no relationship either with clinical features or with treatment issue. We also found no correlation between the cytokines serum concentration and the extent of either angiogenesis or lymphangiogenesis.

Although the extent of lymphangiogenesis seems to be similar in DLBCL and non-neoplastic lymph nodes, the LVD may be a negative predictive factor with regard to the treatment of DLBCL with R-CHOP immunochemotherapy. Although our results do not support the usefulness of serum concentration of VEGF-C, -D, VEGFR-3 and bFGF as surrogate markers of angiogenesis and  lymphangiogenesis  in DLBCL, further studies on this issue would be worthwhile.

The work was supported from a grant of Medical University of Lodz (503/1-093-01/503-01).